Method for detecting and quantifying endogenous wheat DNA sequence

ABSTRACT

A circular DNA is provided comprising endogenous DNA common to both genetically modified wheat and non-genetically modified wheat along with one or more pieces of DNA each having a sequence present specifically in a strain of genetically modified wheat. Also provided is a method for determining a mix rate of genetically modified wheat in a test sample.

This is a divisional of U.S. patent application Ser. No. 11/578,107,filed Aug. 12, 2008, now U.S. Pat. No. 8,030,463 which is the NationalStage of PCT International Application No. PCT/JP2005/006784, filed Apr.6, 2005, and Japanese Application No. JP 2004-115687, filed on Apr. 9,2004, all of which are incorporated herein by reference.

BACKGROUND

The present invention relates to a method for detecting or quantifyingan endogenous DNA sequence of wheat in a test sample, and relates inparticular to an endogenous wheat DNA detection or quantification methodto be used when determining the contamination rate of geneticallymodified wheat contained in food materials or processed foods.

In Japan, 50 or more varieties of genetically modified crops (hereunder“GMOs”) including corn, soy beans and potatoes have passed safetyassessment and been approved for import and sale. At the same time,foods containing GMOs must be labeled as such in accordance with the“Labeling Standard for Genetically Modified Foods established by theMinistry of Agriculture, Forestry and Fisheries based on Article 7,paragraph 1 of the Quality Labeling Standard for Processed Foods andArticle 7, paragraph 1 of the Quality Labeling Standard for Fresh Food”(Notification No. 517 of the Ministry of Agriculture, Forestry andFisheries, Mar. 31, 2000) and the “Enforcement of Ministerial Ordinanceamending in part the Ministerial Ordinance on Food Sanitation LawEnforcement Regulations and Compositional Standards, etc. for Milk andMilk Products” (Notice No. 79 of the Food Sanitation Department,Ministry of Health, Labor and Welfare, Mar. 15, 2001).

In other countries, however, GMOs may in some cases be cultivatedtogether with non-GMOs once the safety evaluation has been completed, orcontamination may occur during the process of distribution afterharvest. Moreover, the makers of food products and the like oftencontract the manufacture of processed foods out to manufacturingcompanies, and even if they stipulate that non-GMOs should be used, ifGMOs are used in the plants of the manufacturing companies, theprocessed foods may become contaminated by small quantities of GMOs.Consequently, in order to fulfill their labeling obligations makers offood products and the like must assess and analyze the final processedfood products to verify that they are not contaminated with GMOs.

Methods of detecting GMOs in test samples of processed foods and theirraw materials, etc. include methods of detecting modified DNA bypolymerase chain reaction (PCR) and methods of detecting modifiedproteins by ELISA, but in the case of processed foods GMOs must bedetected by PCR because the proteins have often been denatured by heator pressure and cannot be detected accurately by ELISA.

Methods of assessment and analysis include the methods described in theJAS Analytical Handbook, Manual of Assessment and Analysis forGenetically Modified Foods, Revised Second Edition and those describedin “Concerning Testing Methods for Foods Modified by Recombinant DNATechnology (Partially Revised)” (Notice No. 0618002 of the FoodSanitation Department, Ministry of Health, Labor and Welfare, Jun. 18,2003). These describe that in the testing and analysis of GMOs, it isnecessary to perform PCR using a primer pair recognizing the endogenousDNA of each agricultural product and to verify that a PCR product of theexpected length is obtained, in order to verify that DNA extracted fromthe test sample can be amplified by PCR. When quantifying a GMOcontained in a test sample, a method is used of measuring the mix rateof the modified crop based on the ratio of recombinant DNA to endogenousDNA that is always present in that crop.

In the case of corn for example, primer pairs have been developed thatrecognize each of the 5 lines of approved GMOs, along with a primer pairthat recognizes the SSIIB gene region of endogenous corn DNA (JASAnalytical Handbook, Manual of Assessment and Analysis for GeneticallyModified Foods, Revised Second Edition, IAA Center for Food Quality,Labeling and Consumer Services). Because this primer pair provides thestandard for amount of endogenous DNA in recombinant DNA detection andquantification, the region of endogenous DNA to be amplified should bepresent in a single copy on the genome.

In “Concerning Testing Methods for Foods Modified by Recombinant DNATechnology (Partially Revised)” (Notice No. 1113001 of the FoodSanitation Department, Ministry of Health, Labor and Welfare, Nov. 13,2003), the amplification products by specific primer pairs targetingendogenous corn or soy bean DNA and recombinant DNA are linked to aplasmid and used as the standard substance. The ratio of number ofcopies of recombinant DNA to number of copies of endogenous DNA can beaccurately determined in a test sample by fixed-time quantitative PCR,by performing PCR using this standard substance.

When there are multiple GMO strains as in the case of corn, aparticularly useful technique is to use a common standard substance tomeasure the contamination rate of each strain, which can be done byusing a standard substance having endogenous DNA and DNA specific toeach strain incorporated into a single circular DNA.

It is generally difficult to obtain genes specific to each strain, butonce these have been incorporated into circular DNA, it is possible toprovide a stable supply of strain-specific DNA by replicating thecircular DNA itself.

SUMMARY

While no genetically modified products of common wheat (Triticumaestivum, hereunder sometimes called simply “wheat”) have yet passedsafety assessment, they are expected to appear on the market in the nearfuture. Consequently, methods for detecting and quantifying endogenouswheat DNA and PCR primer pairs for use in such methods need to bedeveloped in preparation for the distribution of GMO wheat. In terms ofits genome structure and the nucleotide sequence of its genes, however,wheat shares a high degree of homology with other cereals such asbarley, rye and oats. In addition to common wheat (Triticum aestivum),moreover, there is also durum wheat (Triticum durum). Durum wheat sharesa particularly high degree of homology with common wheat since itpossesses the (AA, BB) parts of the common wheat genome (AA, BB, DD), sothe likelihood of false detection is high. There is therefore a need formethods capable of specifically detecting the endogenous DNA of commonwheat without falsely detecting DNA derived from durum wheat and othercereal crops, or in other words for methods which avoid cross-reactionwith other crops.

When there are multiple copies of an endogenous DNA region amplified byPCR, the wheat in the test sample cannot be assayed accurately, so inorder to accurately assay the contamination rate of GMO wheat in a testsample it is desirable that the region of endogenous DNA to be amplifiedbe present in only a single copy on the genome.

Moreover, when assessing GMO contamination by quantitative PCR, it isalso useful in the case of wheat to use a standard substance comprisinga region capable of being amplified by specific primer pairs targetingendogenous gene DNA and recombinant DNA linked on circular DNA.

It is therefore an object of the present invention to specify a partialsequence of wheat DNA (genome) that is present in a single copy andallows specific detection of wheat without cross-reactivity with otherplants in PCR, and to provide primers for amplifying this partialsequence and a method for favorably detecting and assaying endogenousDNA using these primers.

It is another object of the present invention to provide a standardsubstance comprising a region capable of being amplified by specificprimer pairs targeting endogenous gene DNA and recombinant DNA linked oncircular DNA.

The inventors in this case perfected the present invention as a resultof exhaustive research aimed at solving the aforementioned problems whenthey found that the WaxyD gene nontranscribed region, a region of theTaSUT1D gene coding for a sucrose transporter, a region of the CbpIIIgene coding for carboxypeptidase III, the GSS (Genome Survey Sequence)sequence and the Lr1 gene (Leaf rust resistance gene) region are presentin single copies in wheat genome DNA and do not have cross-reactivitywith other plants in PCR, and discovered partial sequences which, whenamplified, allow endogenous wheat DNA sequences to be specificallydetected and assayed.

That is, the present invention relates to:

[1] A method for detecting or assaying endogenous wheat DNA in a testsample by PCR, comprising a step of using a nucleic acid in the sampleor a nucleic acid extracted from the sample as the template to amplifythe nucleic acid of a region comprising at least 80% or more of anucleotide sequence represented by one of SEQ ID NOS:1-7 using a primerpair capable of amplifying that region, and a step of detecting orassaying the amplified nucleic acid;[2] The method according to [1] above, wherein the primer pair isselected from the group consisting of (i) a primer pair consisting of anucleic acid comprising the nucleotide sequence represented by SEQ IDNO:8 and a nucleic acid comprising the nucleotide sequence representedby SEQ ID NO:9, (ii) a primer pair consisting of a nucleic acidcomprising the nucleotide sequence represented by SEQ ID NO:10 and anucleic acid comprising the nucleotide sequence represented by SEQ IDNO:11, (iii) a primer pair consisting of a nucleic acid comprising thenucleotide sequence represented by SEQ ID NO:12 and a nucleic acidcomprising the nucleotide sequence represented by SEQ ID NO:13, (iv) aprimer pair consisting of a nucleic acid comprising the nucleotidesequence represented by SEQ ID NO:14 or 16 and a nucleic acid comprisingthe nucleotide sequence represented by SEQ ID NO:15 or 17, (v) a primerpair consisting of a nucleic acid comprising the nucleotide sequencerepresented by SEQ ID NO:18 and a nucleic acid comprising the nucleotidesequence represented by SEQ ID NO:19, (vi) a primer pair consisting of anucleic acid comprising the nucleotide sequence represented by SEQ IDNO:20 and a nucleic acid comprising the nucleotide sequence representedby SEQ ID NO:21, and (vii) primer pairs consisting of pairs of nucleicacids with each nucleic acid comprising the continuous sequence of atleast 80% of the nucleotide sequence of a nucleic acid in one of theprimer pairs (i) to (vi);[3] A method according to [1] or [2] above wherein the primers in theprimer pair are nucleic acids 15 to 40 nucleotides in length;[4] A primer pair for detecting or assaying wheat in a test sample byPCR, wherein said primer pair is capable of amplifying a regioncomprising at least 80% of a nucleotide sequence represented by any oneof SEQ ID NOS:1 through 7.[5] The primer pair according to [4] above, selected from the groupconsisting of (i) a primer pair consisting of a nucleic acid comprisingthe nucleotide sequence represented by SEQ ID NO:8 and a nucleic acidcomprising the nucleotide sequence represented by SEQ ID NO:9, (ii) aprimer pair consisting of a nucleic acid comprising the nucleotidesequence represented by SEQ ID NO:10 and a nucleic acid comprising thenucleotide sequence represented by SEQ ID NO:11, (iii) a primer pairconsisting of a nucleic acid comprising the nucleotide sequencerepresented by SEQ ID NO:12 and a nucleic acid comprising the nucleotidesequence represented by SEQ ID NO:13, (iv) a primer pair consisting of anucleic acid comprising the nucleotide sequence represented by SEQ IDNO:14 or 16 and a nucleic acid comprising the nucleotide sequencerepresented by SEQ ID NO:15 or 17, (v) a primer pair consisting of anucleic acid comprising the nucleotide sequence represented by SEQ IDNO:18 and a nucleic acid comprising the nucleotide sequence representedby SEQ ID NO:19, (vi) a primer pair consisting of a nucleic acidcomprising the nucleotide sequence represented by SEQ ID NO:20 and anucleic acid comprising the nucleotide sequence represented by SEQ IDNO:21, and (vii) primer pairs consisting of pairs of nucleic acids witheach nucleic acid comprising the continuous sequence of at least 80% ofthe nucleotide sequence of a nucleic acid in one of the primer pairs (i)to (vi);[6] A kit for detecting or assaying an endogenous wheat DNA sequence ina test sample by PCR, comprising a primer pair according to [4] or [5]above;[7] Circular DNA comprising endogenous DNA common to both geneticallymodified wheat and non-genetically modified wheat along with one or morepieces of genetically modified wheat-specific DNA each comprising asequence specific to a strain of genetically modified wheat;[8] Circular DNA comprising DNA comprising a nucleotide sequence havingat least 80% homology with a nucleotide sequence represented by any oneof SEQ ID NOS:1 through 7;[9] Circular DNA comprising a region capable of being amplified by PCRusing a primer pair according to [4] or [5] above;[10] Circular DNA according to [9] or [10] above, further comprising 1or more pieces of DNA each comprising a sequence particular to aspecific strain of genetically modified wheat;[11] A method for determining the contamination rate of geneticallymodified wheat in a test sample, comprising: performing quantitative PCRusing, as templates, the circular DNA described in any one of [7]through [10] above and DNA extracted from the test sample; using theresults of the quantitative PCR for the circular DNA to prepare acalibration curve for determining the number of molecules of templateDNA; using the calibration curve and the results of the quantitative PCRfor the test sample to determine the number of molecules of a partialsequence of an endogenous wheat DNA sequence and the number of moleculesof a partial sequence of a DNA sequence specific to at least one kind ofgenetically modified wheat contained in the test sample; and determiningthe ratio A obtained by dividing the number of molecules of the partialsequence of the DNA sequence specific to the genetically modified wheatby the number of molecules of the partial sequence of the endogenouswheat DNA sequence;[12] The method according to [11] above, further comprising a step ofdetermining the contamination rate of genetically modified wheat in asample by calculating the formula 100×NB using the ratio A and the ratioB obtained by dividing the number of molecules, obtained by quantitativePCR using as the template DNA extracted from standard seeds ofgenetically modified wheat, of a partial sequence of a DNA sequencespecific to a particular strain of genetically modified wheat by thenumber of molecules of a partial sequence of an endogenous wheat DNAsequence;[13] A method according to [11] or [12] above, wherein at least oneprimer pair selected from the primer pairs described in [4] and [5] isused in said quantitative PCR.

The method of the present invention provides accurate information aboutthe presence and amount of wheat in test samples of food materials andprocessed foods, etc., and therefore the PCR primer pairs used in thepresent invention need to specifically detect wheat without anycross-reactions by crops other than wheat such as rice, barley, rye,oats, “minorimugi” barley, corn, soy beans, potatoes, tomatoes,eggplants, foxtail millet, Chinese millet, buckwheat, rapeseed, etc.Moreover, the endogenous DNA region to be amplified by the primer pairof the present invention is preferably a single copy.

If the PCR primer pair is cross-reactive with a crop other than wheat inan assessment method, not only may there be false-positive results forwheat detection but it will be difficult to accurately quantify theendogenous wheat DNA in the test sample. Similarly, endogenous wheat DNAcannot be accurately assayed if the endogenous DNA region is present inmultiple copies. Consequently, such methods and primer pairs cannotaccurately determine the contamination rate of GMO wheat.

The present invention provides a method capable of specificallydetecting or quantifying endogenous wheat DNA in test samples of foodmaterials, processed foods and the like without cross-reacting withother crops, along with PCR primer pairs for use in this method. Thismethod detects or assays by PCR a specific partial sequence of anendogenous DNA sequence which has low homology with grains other thanwheat and which is present in only a single copy on the genome.

Moreover, using the standard substance for detecting GMO wheat providedby the present invention it is possible to accurately determine thecontamination rate of GMO wheat in a test sample for each GMO strain byquantitative PCR.

DESCRIPTION OF DRAWINGS

FIG. 1 shows results for wheat specificity test of the primer pairWx011-5′/3′. Of the lanes, M shows the 100 bp ladder marker, 1 is wheatbrand 1CW, 2 is wheat brand WW, 3 is wheat brand N61, 4 is wheat flower,5 is rice, 6 is barley, 7 is corn, 8 is soy beans, 9 is potatoes, 10 istomatoes, 11 is eggplant, 12 is rye, 13 is Minorimugi, 14 is oats, 15 isfoxtail millet, 16 is Chinese millet, 17 is buckwheat, 18 is rapeseedand 19 is the No Template Control (water).

FIG. 2 shows results for wheat specificity test of the primer pairWx012-5′/3′. Of the lanes, M is the 100 bp ladder marker, 1 is wheatbrand 1 CW, 2 is wheat brand WW, 3 is wheat brand N61, 4 is wheatflower, 5 is rice, 6 is barley, 7 is corn, 8 is soy beans, 9 ispotatoes, 10 is tomatoes, 11 is eggplant, 12 is rye, 13 is Minorimugi,14 is oats, 15 is foxtail millet, 16 is Chinese millet, 17 is buckwheat,18 is rapeseed and 19 is the No Template Control (water).

FIG. 3 shows the designing of the WxS01 probe for Southern hybridizationto estimate the number of copies of Wx012. A shows the nontranscribedregion and B shows the probe.

FIG. 4 shows the restriction enzyme cleavage sites of the WxS01 probe.

FIG. 5 shows the results of Southern hybridization for wheat using theWxS01 probe. Of the lanes, 1 shows the results for WW variety ΔΔ cleavedwith Mbol, 2 for \NW variety ΔΔ cleaved with Mval, 3 for WW variety □□cleaved with EcoT14I, 4 for HRS variety □□ cleaved with Mbol, 5 for HRSvariety □□ cleaved with Mval, 6 for HRS variety □□ cleaved with EcoT14I,and P the results for cleavage with the Positive Control (Wxs01: 300 g).

FIG. 6 shows the results of Southern hybridization for durum wheat. Ofthe lanes, 1 shows the results for durum variety ▾▾ cleaved with Mbol, 2for durum variety ▾▾ cleaved with Mval, and 3 for durum variety ▾▾cleaved with EcoT14I.

FIG. 7 shows the results of a test confirming the wheat specificity ofthe primer pair Cbp014-5′/3′. Of the lanes, M shows the 100 bp laddermarker, 1 the wheat brand 1 CW, 2 the wheat brand WW, 3 the wheat brandN61, 4 wheat flour, 5 rice, 6 barley, 7 corn, 8 soy beans, 9 potatoes,10 tomatoes, 11 eggplant, 12 rye, 13 Minorimugi, 14 oats, 15 foxtailmillet, 16 Chinese millet, 17 buckwheat, 18 rapeseed, and 19 the NoTemplate Control.

FIG. 8 is a calibration curve prepared from the results of quantitativePCR using as templates various concentrations of wheat DNA.

DETAILED DESCRIPTION

The terms used in these specifications are defined below, and thepresent invention is explained in detail.

In these specifications, the term “wheat” indicates common wheat(Triticum aestivum) unless otherwise specified.

The method of the present invention detects as an endogenous DNAsequence of wheat, specific parts of the regions of the WaxyD genenontranscribed region and its 3′ upstream region (SEQ ID NO:16), theTaSUT1D gene (Accession No. AF408845), the CbpIII gene (Accession No.J02817), the Lr1 gene (Accession No. 579983) and the GSS sequence(Accession No. AJ440705) on the wheat genome.

A total of three sets of the Waxy gene are known to exist, one each onthe wheat 4A, 7A and 7D chromosomes (Japanese Patent ApplicationLaid-open No. 2003-284598 and Ainsworth, C. et al., Plant Mol. Biol.1993 April, 22(1):67-82). It is difficult to detect the full lengthWaxyD gene from the wheat genome in a processed food, while the regionof a partial sequence selected at random may be multiple-copy sequence.

The present inventors determined the nucleotide sequence (SEQ ID NO:22)of the nontranscribed region of the WaxyD gene, discovered the existencein that region of a single copy region present only in genome D, andnamed a 101 bp part of this the Wx011 region (SEQ ID NO:2). A 102 bppart was named the Wx012 region (SEQ ID NO:1). In the method of thepresent invention, the WaxyD gene is detected and quantified byamplifying a region comprising at least 80% of the Wx011 region or Wx012region by PCR.

The TaSUT gene is known as a sucrose transporter gene, and the wheat A,B and D chromosomes have been reported to each contain 1 copy of theTaSUT gene with highly homologous nucleotide sequences (Aoki, N. et al.,Plant Molecular Biology 50:453-462, 2002). However, in the presentinvention it was discovered that, of the regions of the genome D TaSUT1Dgene, the sut01 region (SEQ ID NO:3) and sut02 region (SEQ ID NO:4)might be present only in single copies on the D chromosome. Therefore,the present invention also relates to a method for detecting andquantifying the TaSUT1D gene by amplifying a region comprising at least80% of the sut01 region (SEQ ID NO:3) and/or sut02 region (SEQ ID NO:4)by PCR.

Of the regions of the CbpIII gene on the genome the 100 bp CbpIII014region (SEQ ID NO:5) is probably a single copy, and was confirmed tohave almost no cross-reactivity with other plant varieties inqualitative PCR. The present invention therefore relates to a method fordetecting and quantifying the CbpIII gene by amplifying a regioncomprising at least 80% of the CbpIII014 region (SEQ ID NO:5) by PCR.

The wheat GSS region is DNA that has a promoter-like function in genomeanalysis. Of the GSS region, the 111 bp gss01 region (SEQ ID NO:6) isprobably a single copy, and was confirmed to have almost nocross-reactivity with other plant varieties in qualitative PCR. Thepresent invention therefore relates to a method for detecting andassaying the gss01 region by amplifying a region comprising at least 80%of the gss01 (SEQ ID NO:6) region by PCR.

Of the regions of the Lr1 gene in the wheat genome, the 111 bp Lr101region (SEQ ID NO:7) is probably a single copy, and was confirmed tohave almost no cross-reactivity with other plant varieties inqualitative PCR. The present invention therefore relates to a method fordetecting and assaying the Lr1 gene by amplifying a region comprising atleast 80% of the Lr101 region (SEQ ID NO:7) by PCR.

Because the aforementioned Wx011 region, Wx012 region, sut01 region,sut02 region, CbpIII014 region, gss01 region and Lr101 region are allshort (roughly 100-130 bp), they allow endogenous wheat DNA to bedetected and quantified even in processed foods and other samples inwhich the DNA may have become fragmented.

In the specifications of this application, a “region comprising at least80% of a nucleotide sequence represented by any one of SEQ ID NOS:1through 7” is either a shorter region comprising the continuous sequenceof at least 80% of the nucleotide sequence represented by any one of SEQID NOS:1 through 7, or a longer region comprising the nucleotidesequence represented by any one of SEQ ID NOS:1 through 7 along with the5′ end and/or 3′ end nucleotide sequence on the genome, wherein at least80% of the whole constitutes the nucleotide sequence represented by anyone of SEQ ID NOS:1 through 7. Because this region encompasses at least80% of a single copy region, a PCR product of the expected length can beobtained by selecting appropriate primer pairs even if the region isshorter or longer than the nucleotide sequence represented by SEQ IDNOS:1 through 7, allowing endogenous wheat DNA to be detected and/orassayed.

The primer pair used in PCR in the present invention is not particularlylimited as long as it is capable of amplifying a region of at least 80%of the Wx011 region, Wx012 region, sut01 or sut02 region, CbpIII014region, gss01 region or Lr101 region, and can be designed based on thenucleotide sequence of the region to be amplified in accordance with thebasic rules of primer preparation. In this case, care should be taken asto the uniformity of the Tm values of the primers. Each primer should benormally 15 to 40 bp or preferably 15 to 30 bp long.

If the PCR primer pair cross-reacts with a crop other than wheat, notonly may there be false-positive results for wheat detection, but itwill be difficult to accurately quantify the endogenous wheat DNAsequence in the sample. It will also be impossible to accurate assay anendogenous wheat DNA sequence if there are multiple copies of theendogenous DNA sequence. Therefore, such methods and primer pairs cannotaccurately determine the mix rate of GMO wheat.

The method of the present invention provides accurate information aboutthe presence and amount of wheat in test samples of food materials,processed foods, and the like, and therefore the PCR primer pairs usedin the present invention need to specifically detect wheat without anycross-reactions with crops other than wheat such as rice, durum, barley,rye, oats, “minorimugi” barley, corn, soy beans, potatoes, tomatoes,eggplants, foxtail millet, Chinese millet, buckwheat, rapeseed, etc.

Examples of such primer pairs include (i) a primer pair consisting of anucleic acid comprising the nucleotide sequence represented by SEQ IDNO:8 and a nucleic acid comprising the nucleotide sequence representedby SEQ ID NO:9, (ii) a primer pair consisting of a nucleic acidcomprising the nucleotide sequence represented by SEQ ID NO:10 and anucleic acid comprising the nucleotide sequence represented by SEQ IDNO:11, (iii) a primer pair consisting of a nucleic acid comprising thenucleotide sequence represented by SEQ ID NO:12 and a nucleic acidcomprising the nucleotide sequence represented by SEQ ID NO:13, (iv) aprimer pair consisting of a nucleic acid comprising the nucleotidesequence represented by SEQ ID NO:14 or 16 and a nucleic acid comprisingthe nucleotide sequence represented by SEQ ID NO:15 or 17, (v) a primerpair consisting of a nucleic acid comprising the nucleotide sequencerepresented by SEQ ID NO:18 and a nucleic acid comprising the nucleotidesequence represented by SEQ ID NO:19, (vi) a primer pair consisting of anucleic acid comprising the nucleotide sequence represented by SEQ IDNO:20 and a nucleic acid comprising the nucleotide sequence representedby SEQ ID NO:21, and (vii) primer pairs consisting of pairs of nucleicacids with each nucleic acid comprising the continuous sequence of atleast 80% of the nucleotide sequence of a nucleic acid in one of theprimer pairs (i) to (vi) above. These primer pairs specifically amplifythe Wx011 region, Wx012 region, sut01 or sut02 region, CbpIII014 region,gss01 region or Lr101 region without crossing with other crops.

The “primers consisting of nucleic acids each comprising the continuoussequence of at least 80% of the nucleotide sequence of each primer” areprimers each comprising the continuous sequence of at least 80% of oneof the nucleotide sequences represented by SEQ ID NO:8 through 21, whichmay be shifted towards the 5′ end or 3′ end of the genome nucleotidesequence and may be shorter, longer or identical. Consequently, amongthe primer pairs (vii) above, either the forward primer, the reverseprimer or both of them consisting of nucleotide sequences represented bySEQ ID NOS:8 through 21 may be modified according to the aforementionedconditions. However, because these primers comprise at least 80% ofnucleotide sequences represented by SEQ ID NOS:8 through 21, like theprimer pairs (i) through (vi) above, they can specifically amplify theWx011 region, Wx012 region, sut01 or sut02 region, CbpIII014 region,gss01 region or Lr101 region without cross-reacting with other crops.

The test samples used in the present invention are food materials orprocessed foods that contain or may contain wheat, including for exampleraw wheat seeds, dried seeds, wheat flour, mixed flour and other rawfoodstuffs and intermediate processed foodstuffs, as well as processedfoods such as bread and noodles. These food materials and products arenot limited to human foods but also include pet foods and feed. Cropsother than wheat include all crops used as food materials and rawfoodstuffs, such as those mentioned above.

Nucleic acids can be extracted from this sample either as is or after ithas been pulverized, or else after it has been washed, dried andpulverized. The nucleic acids extracted from the test sample and used inanalysis are normally DNA. The DNA may be extracted by any known method,and may be extracted using one of the many DNA extraction kits currentlyon the market. For example, DNA can be extracted from the test sampleusing a DNeasy Plant Maxi Kit (QIAGEN) in accordance with the methodsdescribed in the JAS Analytical Handbook, Manual of Assessment andAnalysis for Genetically Modified Foods, Revised Second Edition (IAACenter for Food Quality, Labeling and Consumer Services). Theconcentration of the extracted DNA is measured by absorption, and it ispreferably used after having been diluted to a suitable concentrationfor PCR.

In the method of the present invention, PCR can be performed inaccordance with ordinary methods taking into consideration the primersand DNA polymerase used. The PCR buffer, dNTP, MgCl₂ and other reagentsmay be prepared, or a commercial PCR kit may be used. One or two or morepairs of the aforementioned primers may be used in PCR. The PCRconditions may be for example 40 cycles of a cycle of 30 seconds at 95°C., 30 seconds at 63° C. and 30 seconds at 72° C., followed by 7 minutesat 72° C. for the final reaction, but these conditions can be changed asappropriate taking into consideration the Tm of the primers used, thelength of the region to be amplified, the concentration of template DNAand the like.

The amplified nucleic acid (PCR product) can be detected using anymethod capable of identifying a specific DNA fragment, such as agarosegel electrophoresis, acrylamide gel electrophoresis, capillaryelectrophoresis, hybridization, immunological methods or the like. Ingeneral, the PCR product is electrophoresed and identified based on theelectrophoresis pattern, but detection can also be accomplished byidentifying a band produced by electrophoresis using 0.8% agarose gelcontaining ethidium bromide.

The present invention encompasses the primer pairs used in theaforementioned detection or assay method and a kit containing theseprimer pairs. The primers can be manufactured by ordinary methods. Inaddition to the primer pairs, the kit may include other reagents such asdNTP, MgCl₂, Taq DNA polymerase and other polymerases, buffer (such asTris-HCl), glycerol, DMSO, positive control DNA, negative control DNA,distilled water and the like. These reagents may be packagedindividually within the kit, or two or more reagents can be providedmixed with one another. The concentrations of the various reagents inthe kit are not particularly limited as long as they are such as toallow the PCR of the present invention. The kit may consist only of theprimer reagents or may also include desirable PCR conditions and otherinformation.

The present invention also provides a standard substance useful formeasuring the contamination rate of GMO wheat by quantitative PCR. Thisstandard substance comprises endogenous DNA common to both non-GMO wheatand GMO wheat along with one or more pieces of GMO wheat-specific DNAlinked on a single circular DNA.

The standard substance of the present invention may be circular DNAcomprising, as the endogenous DNA, DNA consisting of a nucleotidesequence having at least 80% homology with any one of the nucleotidesequences represented by SEQ ID NOS1 through 7.

It may also be circular DNA comprising, as the endogenous DNA, a regioncapable of being amplified by a primer pair selected from the groupconsisting of (i) a primer pair consisting of a nucleic acid comprisingthe nucleotide sequence represented by SEQ ID NO:8 and a nucleic acidcomprising the nucleotide sequence represented by SEQ ID NO:9, (ii) aprimer pair consisting of a nucleic acid comprising the nucleotidesequence represented by SEQ ID NO:10 and a nucleic acid comprising thenucleotide sequence represented by SEQ ID NO:11, (iii) a primer pairconsisting of a nucleic acid comprising the nucleotide sequencerepresented by SEQ ID NO:12 and a nucleic acid comprising the nucleotidesequence represented by SEQ ID NO:13, (iv) a primer pair consisting of anucleic acid comprising the nucleotide sequence represented by SEQ IDNO:14 or 16 and a nucleic acid comprising the nucleotide sequencerepresented by SEQ ID NO:15 or 17, (v) a primer pair consisting of anucleic acid comprising the nucleotide sequence represented by SEQ IDNO:18 and a nucleic acid comprising the nucleotide sequence representedby SEQ ID NO:19, (vi) a primer pair consisting of a nucleic acidcomprising the nucleotide sequence represented by SEQ ID NO:20 and anucleic acid comprising the nucleotide sequence represented by SEQ IDNO:21, and (vii) primer pairs consisting of pairs of nucleic acids witheach nucleic acid comprising the continuous sequence of at least 80% ofthe nucleotide sequence of a nucleic acid in one of the primer pairs (i)to (vi) above.

The circular DNA used for the standard substance is not particularlylimited as long as it allows insertion of endogenous DNA and GMO wheatstrain-specific DNA, but for example a pBR vector (pBR322, pBR328,etc.), pUC vector (pUC19, pUC18, etc.) or A-phage vector (λgt10, λgt11,etc.) or a commercial vector comprising one of these with modificationsor the like can be used.

When detecting GMO wheat, it is necessary not only to amplify and detectan exogenous DNA sequence inserted into the normal wheat genome bygenetic recombination, but to amplify a region including the endogenoussequences upstream and downstream from the exogenous DNA sequence.Because the identical exogenous DNA sequence may be inserted into othercrops to prepare GMO crops, if only the exogenous DNA sequence isdetected it will not be clear whether it is derived from GMO wheat orfrom another genetically modified crop. Consequently, primers fordetecting GMO strain-specific sequences need to be primers capable ofamplifying regions comprising the endogenous sequences upstream anddownstream from the exogenous DNA sequences inserted into each strain ofGMO wheat. These primers are prepared according to the methods describedwith respect to soy beans for example (Wurz, A. et al., 2^(nd) Statusreport: BgVV, BgVV-Heft, 1/199797, 118 or Kopell, E. et al., Mitt.Gebiete Levensm, Hyg., 88, 164, etc.), or analogous methods. A DNAsequence capable of being amplified by these primers is selected as theGMO wheat strain-specific sequence to be inserted into the standardsubstance.

Once the endogenous wheat DNA and GMO wheat-specific DNA to be insertedinto the standard substance have been determined, PCR is performed usinga normal wheat genome or GMO wheat genome as the template to clone theendogenous DNA and GMO wheat-specific DNA, and the cloned DNA fragmentsand the cloning site of the aforementioned circular DNA are cleaved withthe same restriction enzyme to link the DNA fragments to the cleavedsite of the circular DNA. A known restriction enzyme can be selected asappropriate, and for example EcoRI, SpeI, EcoRV, SmaI, SacI, NotI,HindIII, XhoI or the like can be used.

Calibration curves can be derived for the partial sequences of theendogenous wheat DNA sequence and GMO-specific DNA sequence by preparinga dilution series of two or more concentrations of a solution comprisingthe resulting standard substance, and subjecting each to quantitativePCR. Moreover, the standard substance of the present invention can beused as a positive control for the endogenous wheat DNA sequence orGMO-specific DNA sequence in qualitative PCR.

The present invention encompasses a method for determining thecontamination rate of GMO wheat in a test sample by PCR using theaforementioned standard substance.

In this method, quantitative PCR is performed using as templates theaforementioned standard substance and DNA extracted from the testsample, and a calibration curve for determining the number of moleculesof template DNA is prepared using the results of quantitative PCR forthe standard substance.

In quantitative PCR, the Ct value is used as data. The Ct value is thecycle number (threshold cycle) at which a specific amount ofamplification product is reached as amplification becomes exponentialwhen changes in the amount of amplified product are followed over timein quantitative PCR. The aforementioned calibration curve can be used toconvert this Ct value into the initial number of molecules of DNA(number of molecules of template DNA) contained in the test samplebefore PCR.

The calibration curve can be prepared according to known methods orsimilar methods with for example the Ct value plotted on the verticalaxis and the logarithm of the number of molecules of standard substancein the dilution series on the horizontal axis. For example, it can beprepared by preparing a dilution series containing variousconcentrations of the standard substance and deriving the Ct values foreach following a fixed period of quantitative PCR.

The initial number of molecules of a partial sequence of the endogenouswheat DNA sequence contained in the test sample and the number ofmolecules of a partial sequence of a DNA sequence specific togenetically modified wheat can be derived using the aforementionedcalibration curve from the Ct values or in other words from the resultsof quantitative PCR performed on the test sample.

The resulting number of molecules of the partial sequence of a DNAsequence specific to genetically modified wheat can be divided by thenumber of molecules of the partial sequence of an endogenous wheat DNAsequence to obtain ratio A, while the number of molecules of a partialsequence of a DNA sequence specific to each strain of GMO wheat obtainby quantitative PCR using standard seeds of genetically modified wheatcan be divided by the number molecules of the partial sequence of anendogenous wheat DNA sequence to obtain ratio B, and the contaminationrate of genetically modified wheat in the test sample can be determinedby calculating the formula 100×A/B. This ratio B is called the “internalstandard ratio” in the JAS Analytical Handbook, Manual of Assessment andAnalysis for Genetically Modified Foods, Revised Second Edition, IAACenter for Food Quality, Labeling and Consumer Services, and is the(recombinant gene)/(endogenous gene) ratio in DNA extracted from seedsof each pure GM strain. The internal standard ratio is constant in eachstrain of recombinant seeds.

The PCR steps in the method of the present invention for determining theGMO wheat contamination rate can be performed either simultaneously orseparately. When the PCR steps are performed separately, the conditionsshould be such as to produce roughly the same nucleic acid amplificationefficiency as in the PCR for determining the calibration curve. Anexample of such conditions would be the same temperatures and cycles asin the PCR for preparing the calibration curve.

Example 1 WaxyD Gene Detection

Detection was accomplished by amplifying the 101 bp Wx011 region (SEQ IDNO:2) and 102 bp Wx 012 region (SEQ ID NO:1), which are nontranscribedregions of the WaxyD gene.

[1] Primer Design

The primers were designed using the primer design software PrimerExpress (Applied Biosystems). The primers were designed in strictaccordance with the basic rules of primer preparation, so that inaddition to obtaining a uniform Tm value for each primer, theamplification product of PCR would be about 100 to 150 bp in order toallow detection in processed foods with DNA fragmentation, and thenucleotide length of each primer would be 18 to 25 bp. The 5′ primerWx011-5′ (SEQ ID NO:8) and 3′ primer Wx011-3′ (SEQ ID NO:9) and the 5′primer Wx012-5′ (SEQ ID NO:10) and 3′ primer Wx012-3′ (SEQ ID NO:11)were obtained as a result.

[2] DNA Extraction

For the PCR template DNA samples, DNA extracted from 2 brands (1CW, WW)and 4 varieties (including N61) of wheat and commercial wheat flour(Nisshin Flour Milling Co., Ltd. “Kameriya”) was used for the wheatsamples, while DNA extracted from rice, corn, foxtail millet, Chinesemillet, buckwheat, 2 varieties of barley, rye, oats, soy beans,rapeseed, tomatoes, eggplant, and 1 brand (CAD) and 4 varieties(hereunder called “durum varieties A-D) of durum wheat were used as thecomparative samples.

The wheat and other plant samples were washed with 1% SDS (Wako PureChemical Ind.), rinsed with distilled water and thoroughly dried, andthen finely pulverized using a multi-bead shocker (Yasui Machines). DNAwas extracted from 1 g of each pulverized sample using a DNeasy PlantMaxi Kit (QIAGEN) in accordance with the DNA extraction protocols forcorn described in the JAS Analytical Handbook, Manual of Assessment andAnalysis for Genetically Modified Foods, Revised Second Edition, IAACenter for Food Quality, Labeling and Consumer Services. For the durumwheat, 4 kernels of each variety were selected randomly and DNA wasextracted from each kernel using the aforementioned kit in accordancewith the attached protocols. The concentration of the extracted DNA wasmeasured from the absorption, and part of it was diluted 10 ng/μL withpure water and used as the template DNA sample liquid in the PCRreaction.

[3] PCR Reaction and Electrophoresis

The PCR reaction liquids were prepared as follows. That is, 2.5 μL ofDNA sample liquid prepared to 10 ng/μL was added to a liquid comprisingPCR buffer (PCR buffer II, Applied Biosystems), 200 μmol/L dNTP, 1.5mmol/L MgCl₂, 0.5 μmol/L 5′ and 3′ primers and 0.625 units of Taq DNApolymerase (Ampli Taq Gold, Applied Biosystems), for a total volume of25 μL.

Using a GeneAmp PCR System 9600 (Applied Biosystems) as the PCRamplifier, the reaction conditions were set as follows. The temperaturewas maintained at 95° C. for 10 minutes to initiate the reaction, andPCR amplification was performed in 40 cycles of a cycle consisting of 30seconds at 95° C., 30 seconds at 63° C. and 30 seconds at 72° C. Thereaction liquid was then maintained for 7 minutes at 72° C. for thefinal reaction, stored at 4° C., and used as the PCR amplificationreaction liquid.

The PCR amplification reaction liquid was electrophoresed on 0.8%agarose gel containing ethidium bromide. The results of the detectiontests for wheat and other crops are shown in FIG. 1 for Wx011-5Wx011-3′and in FIG. 2 for Wx012-5′/Wx012-3′.

Using either the primer pair Wx011-5′/Wx011-3′ or the primer pairWx012-5′/Wx012-3′, a single band of the expected size was detected inthe wheat sample (Lanes 1-4), but this band is not seen in the non-wheatlanes. This shows that the Wx011 region or Wx012 region of endogenouswheat DNA can be detected without crossing from other crops using theWx011-5′/Wx011-3′ or Wx012-5′/Wx012-3′ primer pair.

The results for wheat and durum wheat using the Wx012-5′/Wx012-3′ primerpair are shown in Table 1 below. These results confirm that wheat anddurum wheat can be detected without crossing using the Wx012-5/Wx012-3′primer pair.

TABLE 1 Detection primer Lane No. Template DNA wx012 5′/3′ 1 Durumvariety A − 2 Durum variety B − 3 Durum variety C − 4 Durum variety D −5 Durum (CAD) − N No template control (water) − P Positive control(wheat) + +: Amplification band of optimum size detected −:Amplification band not detected

Example 2 Confirmation of Number of Copies of Wx012 Region

Southern hybridization was performed under the following conditions toconfirm the number of copies of Wx012.

DNA extracted from 2 varieties of wheat was used as the samples. DNA wasextracted as in Example 1.

A Gene Images Alkphos Direct Labeling and Detection System (AmershamBiosciences) was used for hybridization and detection. The reagents,buffers and the like described in the kit protocols were used.

[1] Probe Design

The length of a probe for obtaining good sensitivity in Southernhybridization using this kit is 300 bp or more. However, Wx012 is 102 bpin length, too short for Southern hybridization. Moreover the wheatgenome is large, 1.7×10¹⁰ bp, so in order to adequately enhancedetection sensitivity a 444 bp WxS01 probe comprising the Wx012 regionwas designed and prepared by PCR using the 5′ primer WxS01-5′ (SEQ IDNO:23) and 3′ primer Wx012-3′ (SEQ ID NO:11). The primers for obtainingWxS01 were designed using Genetyx Win, in accordance with the basicrules of primer preparation so as to maintain a uniform Tm value foreach primer. An outline of the primer design is shown in FIG. 3.

[2] Selection of Restriction Enzymes

For the restriction enzymes, enzymes were selected on the condition thatthey did not cleave the target region and were not affected bymethylation, and that the location of a cleavage site comprising thetarget was known. As a result, Mbol, Mval and EcoT14I were selected asbeing capable of cleaving both sides of the target region in a singleenzyme reaction. The respective cleavage sites and fragment sizes areshown in FIG. 4. The restriction enzyme sites shown in FIG. 3 arelocations on the nucleotide sequence represented by SEQ ID NO:22.

[3] Southern Hybridization of Wheat

The DNA was reacted for 15 hours at 37° C. with the aforementionedrestriction enzymes. Following this reaction the product was subjectedto phenol-chloroform treatment and ethanol sedimentation, and thendissolved in TE solution. The resulting DNA solution was electrophoresedwith 1.6% agarose gel (LO3, TaKaRa Bio) using a TAE solution as theelectrophoresis solution. Next, the DNA in the gel was transcribedovernight to a membrane (HyperBondN⁺, Amersham Pharmacia) using a 20×SSCsolution.

Hybridization was performed at 55° C. using the hybridization bufferincluded in the kit. The WxS01 probe was adjusted to a concentration of20 ng/ml and reacted overnight. This was washed for 20 minutes at 55° C.using the primary washing liquid and for 10 minutes at room temperatureusing the secondary washing liquid. After being washed, it was left for3 minutes on the membrane on which the detection enzyme reaction hadbeen initiated, and then wrapped in saran wrap after careful removal ofthe detection liquid. This was developed for 1 hour in a dark room withphotosensitive film (Hyper film, Amersham Pharmacia), and the bands wereevaluated.

As a result, 2 bands were detected after cleavage with Mbol, 3 aftercleavage with Mval, and 3 or 6 after cleavage with EcoT14I (FIG. 5).Since it has been reported that there are 2 copies of the Waxy gene ongenome A and 1 copy on genome D (Ainsworth, C. et al., Plant Mol. Biol.1993 April, 22(1):67-82), this confirms that the region with which theWxS01 probe hybridizes is present in 2 copies on genome A and 1 copy ongenome D.

[4] Southern Hybridization of Durum Wheat

Southern hybridization was performed as in [3] above using as thetemplate durum wheat, which does not include a genome D.

As a result, 1 band was detected after cleavage with Mbol, 2 aftercleavage with Mval and 3 after cleavage with EcoT14I. However, of the 3bands produced by EcoT14I one was thicker than the other two,representing a fusion of two bands, so it was determined that at most 4bands were detected in durum wheat in this test (FIG. 6). These resultsmatch [3] above, which shows that 2 copies of WxS01 are present ongenome A. It was also confirmed in Example 1 that the Wx012 region ofendogenous DNA is present only on genome D. These results confirm thatthere is only one copy of the Wx012 region in the wheat genome.

Example 3 Confirming Suitability of Wx012 by Quantitative PCR

Next, it was confirmed that Wx012 meets the necessary conditions as anendogenous sequence for detection even in quantitative PCR.

[1] TaqMan Probe Design

This was designed using the primer and probe design software PrimerExpress (Applied Biosystems Japan). An appropriate probe was selected bychecking the conditions for probe selection described in the softwareprotocols. The nucleotide sequence of the designed probe is representedby SEQ ID NO:42.

[2] Test Samples

DNA extracted from 12 plants other than wheat (rice, corn, foxtailmillet, Chinese millet, buckwheat, barley, rye, oats, soy beans,rapeseed, garbanzo beans, kidney beans), 4 typical varieties of durumwheat and 19 typical varieties of strong, medium-strength andlow-strength wheat was used for the PCR template DNA samples.

[3] DNA Extraction

The wheat and other plant samples were each washed with 1% SDS (WakoPure Chemical Ind.), rinsed with distilled water, thoroughly dried andfine pulverized using a multi-bead shocker (Yasui Machine). DNA wasextracted from 1 g of each of the resulting powdered grain samples usinga DNeasy Plant Maxi kit (Qiagen) in accordance with the corn DNAextraction protocols described in official methods. In the case of thedurum wheat, 4 kernels of each variety were selected randomly, and DNAwas extracted from each kernel in accordance with the protocols of theDNeasy Plant Mini Kit (Qiagen). In the case of 4 varieties of wheat, DNAwas extracted using a Genomic-tip 20/G (Qiagen) and the CTAB method inaccordance with the protocols described in official methods, and alsowith a DNeasy Plant Mini Kit (Qiagen) in accordance with the attachedprotocols. The concentration of the extracted DNA was measured byabsorption, and part was diluted to 20 ng/μL with pure water and used asthe template DNA sample liquid for the PCR reaction.

[4] Quantitative PCR Reaction

Quantitative PCR was performed using an ABI7700 (Applied Biosystems).

A 2-point or 3-point parallel analysis was performed on each sample.Each reaction was performed using a system of 25 μL per well.

The PCR reaction liquid was prepared as follows. Solutions of the TaqMan probe, 5′ primer and 3′ primer were diluted with pure water to 2 μM,5 μM and 5 μM, and a solution of each of these and pure water mixed inproportions of 1:1:1:1 was used as the Primer-Probe Mix solution.

The necessary quantity of the master mix was prepared by mixing TaqManUniversal Master Mix (Applied Biosystems) and the Primer-Probe Mixsolution at a ratio of 1.25:1. 72 μL of the master mix was dispensed foreach template DNA, and 8 μL of each template DNA prepared to 20 ng/mLwas added and thoroughly mixed. 25 μL of this mixture was dispensed tothe designated wells on a 96-well plate, 3 wells per sample.

The reaction conditions were set as follows. The temperature wasmaintained at 50° C. for 2 minutes and then 95° C. for 10 minutes toinitiate the reaction, followed by an amplification reaction of 40cycles of a cycle of 30 seconds at 95° C. and 1 minute at 59° C., afterwhich the temperature was maintained at 50° C. for 4 minutes.

[5] Confirming Wheat Specificity

Using DNA of 19 varieties of wheat and 12 other plants, DNA extractedfrom 4 varieties of durum wheat and DNA extracted from 12 plants otherthan wheat as templates, each sample was applied to 3 wells andquantitative PCR was performed.

As a result of quantitative PCR using the Wx012-5′/3′ primers and TaqManprobe Wx012-T, no amplification curve was detected for NTC, but goodamplification curves were obtained for the 19 varieties of wheat DNA.Amplification was not detected in the case of the DNA from 12 types ofplants other than wheat or the DNA from 4 varieties of durum wheat.

[6] Confirming Linearity of Calibration Curve

Wheat DNA was prepared to 300 ng/μL, 150 ng/μL, 75 μL, 30 μL, 10 ng/μL,4 ng/μL, 1 ng/μL and 0.1 ng/μL, and used as the standard template inquantitative PCR. A calibration curve was derived from the resultsaccording to the JAS Analytical Handbook, and the correlationcoefficient was confirmed.

The resulting calibration curve is shown in FIG. 8. When wheat DNAprepared to 1-300 ng/μL or 0.1-75 ng/μL was applied as standard templateDNA, a highly linear calibration curve was obtained, indicating thatthese can be used in quantitative testing.

Example 4 TaSUT1D Gene Detection

The sut01 region (SEQ ID NO:3) of the TaSUT1D gene and the sut02 region(SEQ ID NO:4) consisting of the described nucleotide sequence wereamplified by PCR.

The primer pairs were designed as in the case of WaxyD above, and the 5′primer sut01-5′ (SEQ ID NO:12) and 3′ primer sut01-3′ (SEQ ID NO:13) andthe 5′ primer sut02-5′ (SEQ ID NO:14) and 3′ primer sut02-3′ (SEQ IDNO:15) were used in PCR testing. The results are shown in Table 2.

TABLE 2 TaSUT detection primers Template DNA sut01-5′/3′ sut02-5′/3′Wheat + + Rice − − Corn − − Soy beans − − Rye − − Oats − − Barley − −Rapeseed − − Foxtail millet − − Chinese millet − − Durum Brand E − − NTC(water) − − +: Amplification band of optimum size detected −:Amplification band not detected NTC: No Template Control (water)

A single band (131 bp or 101 bp) of the anticipated size was detectedusing either primer pair in the case of wheat, while no band wasdetected in the case of other grains and durum wheat (genomeconfiguration AaBb). When the same test was performed using multiplevarieties of wheat, the anticipated single band was detected in each.

It has already been shown that the region from which these primer pairswere designed (Accession No. AF408845, 3924-4397; 474 bp) is present inone copy each in genomes A, B and D. The amplified regions sut01 andsut02 of sut01-573′ and sut02-5′/3′ are specific to wheat (genomeconfiguration AaBbDd) in qualitative PCR and are not detected in durumwheat (genome configuration AaBb), confirming that the sut01 region andsut02 region are probably present in only one copy in genome D. Thisconfirms that endogenous wheat DNA (TaSUT1D gene) can be detected usingthe primer pair sut01-5′/3′ or sut02-5′/3′ without crossing from othercrops.

Example 5 Detection of CbpIII Gene

The CbpIII014 region (SEQ ID NO:5) of the CbpIII gene was amplified byPCR.

The primer pair was designed as in the case of WaxyD above, and PCRtesting was performed using the 5′ primer cbp014-6′ (SEQ ID NO:16) and3′ primer cbp014-3′ (SEQ ID NO:17). The results are shown in FIG. 7. Asingle band of the anticipated size (101 bp) was detected in the wheatlanes (Lanes 1-4), but no band was detected in the non-wheat lanes. Thisconfirms that endogenous wheat DNA (Cbp gene) can be detected using theprimer pair cbp014-5′/cbp014-3′ without crossing from other crops.

Example 6 Detection of GSS Sequence

The gss01 region (SEQ ID NO:6), gss02 region (SEQ ID NO:30) and gss03region (SEQ ID NO:31) of the GSS sequence were amplified by PCR.

The primer pairs were designed as in the case of WaxyD, and PCR wasperformed using the gss01-5′ (SEQ ID NO:18) and gss01-03′ (SEQ ID NO:19)primers as the primer pair for the gss01 region, the gss02-5′ (SEQ IDNO:34) and gss02-3′ (SEQ ID NO:35) primers as the primer pair for thegss02 region, and the gss03-5′ (SEQ ID NO:36) and gss03-3′ (SEQ IDNO:37) primers as the primer pair for the gss03 region. The results areshown in Table 3.

[Table 3]

TABLE 3 Confirmation of specificity of GSS detection primers GSSdetection primers Template DNA gss01-5′/3′ gss02-5′/3′ gss03-5′/3′Wheat + + + Durum wheat − Extra + Rice − − − Barley − − − Corn − + − Soybeans − − − Buckwheat − − − Oats − Rye − Chinese millet − Foxtail millet− Rapeseed − NTC − − − +: Amplification band of optimum size detected −:Amplification band not detected Extra: Band of size different fromoptimum size detected NTC: No Template Control (water)

In the case of wheat a single band of the anticipated size was detectedusing all three primer pairs. Some crossing by durum wheat and cornoccurred with the gss02 and gss03 primer pairs, but no such crossingoccurred with the gss01 primers, confirming that the gss01 primers arecapable of detecting the endogenous wheat gene.

Example 7

The Lr101 region (SEQ ID NO:7), Lr102 region (SEQ ID NO:32) and Lr103region (SEQ ID NO:33) of the Lr1 sequence were amplified by PCR.

The primer pairs were designed as in the case of WaxyD, and PCR wasperformed using the pair of primers represented by SEQ ID NO:20 and SEQID NO:21 for the Lr101 region, the pair of primers represented by SEQ IDNO:38 and SEQ ID NO:39 for the Lr102 region and the pair of primersrepresented by SEQ ID NO:40 and SEQ ID NO:41 for the Lr103 region. Theresults are shown in Table 4.

TABLE 4 Confirmation of specificity of Lr1 detection primers Lr1detection primers Template DNA Lr101-5′/3′ Lr102-5′/3′ Lr103-5′/3′Wheat + + + Durum wheat − + Extra Rice − − − Barley − − − Corn − − − Soybeans − − − Buckwheat − − − Oats − Rye − Chinese millet − Foxtail millet− Rapeseed − NTC − − − +: Amplification band of optimum size detected −:No amplification band detected Extra: Band of different size fromoptimum size detected NTC: No Template Control (water)

In the case of wheat a single band of the anticipated size was detectedusing all three primer pairs. Some crossing by durum wheat occurred withthe Lr102 and Lr103 primer pairs, but no such crossing occurred with theLr101 primers, confirming that the Lr101 primers are capable ofdetecting the endogenous wheat gene.

COMPARATIVE EXAMPLE

Detection tests for endogenous wheat DNA were performed by PCR using (1)a 5′ primer Cbp013-5′ (SEQ ID NO:24)/3′ primer Cbp013-3′ (SEQ ID NO:25)primer pair designed based on the nucleotide sequence of the CbpIIIgene, (2) a 5′ primer TthV011-5′ (SEQ ID NO:26)/3′ primer TthV011-3′(SEQ ID NO:27) primer pair designed based on the nucleotide sequence ofthe TthV gene (Castagnaro A. et al., J. Mol. Biol. 1992 Apr. 20,224(4):1003-9), and a 5′ primer TthV012-5′ (SEQ ID NO:28)/3′ primerTthV012-3′ (SEQ ID NO:29) primer pair designed based on the nucleotidesequence of the TthV gene similar to (3).

DNA extraction, PCR, electrophoresis and the like were performed as inthe aforementioned examples.

Using primer pair (1), a single band of the anticipated size wasdetected from wheat, but the band was also confirmed from rice, barley,corn, rye, “minorimugi” barley, oats and foxtail millet, indicatingcrossing by multiple crops. Using primer pair (2), a single band of theanticipated size was detected from wheat, but there was crossing frombarley. Using primer pair (3), a single band of the anticipated size wasdetected from wheat, but there was crossing from rye and “minorimugi” (atype of barley). The results are shown in Table 5 below.

TABLE 5 CbpIII primers TthV primers Template DNA Cbp013-5′/3′TthV011-5′/3′ TthV012-5′/3′ Wheat + + + Rice Extra − − Barley Extra + −Corn Extra − − Soy beans − − − Potatoes − Tomatoes − − − Eggplant − − −Rye Extra + Barley Extra + (“Minorimugi”) Oats Extra − Foxtail milletExtra − Chinese millet − − Buckwheat − − Rapeseed − − NTC − − +:Amplification band of optimum size detected −: No amplification banddetected NTC: No Template Control (water) blank: not tested

We claim:
 1. An isolated circular DNA comprising the nucleotide sequenceof SEQ ID NO:4 capable of being amplified by PCR using a primer paircomprising a first primer comprising the nucleotide sequence of SEQ IDNO:14 and a second primer comprising the nucleotide sequence of SEQ IDNO:15.
 2. The circular DNA according to claim 1, further comprising oneor more pieces of DNA each comprising a sequence particular to a strainof genetically modified wheat.
 3. A method for determining a mix rate ofgenetically modified wheat in a test sample, comprising: performingquantitative PCR using, as templates, DNA extracted from the test sampleand a circular DNA comprising the nucleotide sequence of SEQ ID No:4capable of being amplified by PCR using a primer pair comprising a firstprimer comprising the nucleotide sequence of SEQ ID NO:14 and a secondprimer comprising the nucleotide sequence of SEQ ID NO:15, preparing acalibration curve for determining the number of molecules of templateDNA, using the results of the quantitative PCR for the circular DNA,determining the number of molecules having a partial sequence of anendogenous wheat DNA sequence and the number of molecules having apartial sequence of a DNA sequence present specifically in at least onekind of genetically modified wheat contained in the test sample, usingthe calibration curve and the results of the quantitative PCR for thetest sample, and determining a ratio A obtained by dividing the numberof molecules having a partial sequence of a DNA sequence presentspecifically in the genetically modified wheat by the number ofmolecules having a partial sequence of an endogenous wheat DNA sequence.4. The method according to claim 3, further comprising: determining themix rate of genetically modified wheat in a sample by calculating aformula 100×A/B using the ratio A and a ratio B obtained by dividing thenumber of molecules, obtained by quantitative PCR using as template DNAextracted from standard seeds of genetically modified wheat, having apartial sequence of a DNA sequence present specifically in a particularstrain of genetically modified wheat by the number of molecules having apartial sequence of an endogenous wheat DNA sequence.